The immunopathogenesis of Entamoeba histolytica

Experimental Parasitology 126 (2010) 366–380

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The immunopathogenesis of Entamoeba histolytica Leanne Mortimer, Kris Chadee * Faculty of Medicine, Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1

a r t i c l e

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Article history: Received 29 October 2009 Received in revised form 8 March 2010 Accepted 12 March 2010 Available online 19 March 2010 Keywords: Amebiasis Amebic colitis Amebic liver abscess Commensal microorgansim Dysentery Entamoeba histolytica Pathogenesis Immunity Immunopathogenesis Immune response Mucosal immune response

a b s t r a c t Amebiasis is the disease caused by the enteric dwelling protozoan parasite Entamoeba histolytica. The WHO considers amebiasis as one of the major health problems in developing countries; it is surpassed by only malaria and schistosomiasis for death caused by parasitic infection. E. histolytica primarily lives in the colon as a harmless commensal, but is capable of causing devastating dysentery, colitis and liver abscess. What triggers the switch to a pathogenic phenotype and the onset of disease is unknown. We are becoming increasingly aware of the complexity of the host–parasite interaction. During chronic stages of amebiasis, the host develops an immune response that is incapable of eliminating tissue resident parasites, while the parasite actively immunosuppresses the host. However, most individuals with symptomatic infections succumb only to an episode of dysentery. Why most halt invasion and a minority progress to chronic disease remains poorly understood. This review presents a current understanding of the immune processes that shape the outcome of E. histolytica infections during its different stages. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction The enteric dwelling protozoan parasite, Entamoeba histolytica is the causative agent of amebiasis. Although primarily a disease of under developed countries, amebiasis may be found in travelers who have returned from endemic areas. Collectively, there are about 100 million annual cases of amebic dysentery, colitis and liver abscess resulting in 100,000 deaths. According to the WHO, amebiasis is the third leading cause of death due to parasitic disease (WHO, 1997). E. histolytica is acquired when infective cysts are ingested through contaminated food or water. Excystation releases trophozoites into the terminal ileum and from there parasites migrate to the colon where they colonize the mucus layer via binding to host mucin oligosaccharides with the ameba surface adhesin, the Galactose/N-acetyl Galactosamine inhibitable lectin (Gal-lectin; Chadee et al., 1987). Establishment in the mucus layer is a pre-requisite for disease, though the majority of amebic infections do not harm the host. Usually, trophozoites remain in the lumen as commensals, multiplying via binary fission and satisfying their energetic needs by ingesting resident microflora and nutrients from the host. Some parasites undergo encystment in the descending colon, resulting in passage of mature infective cysts

* Corresponding author. Fax: +1 403 270 2772. E-mail address: [email protected] (K. Chadee). 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.03.005

in the stool and perpetuation of the life cycle through fecal-oral spread. In 90% of cases amebic infections are asymptomatic and self-limiting (Haque et al., 2003). For unknown reasons, E. histolytica is capable of taking on a pathogenic phenotype. Disease occurs when trophozoites disrupt the mucosal barrier and penetrate the underlying tissue where they secrete enzymes which breakdown extracellular matrix, destroy cells and phagocytose cellular debris. After invading the mucosa and submucosa, trophozoites may enter portal circulation and disperse to the liver and other soft organs. Amebiasis manifests in multiple forms and is broadly categorized based on the site of infection as either intestinal or extraintestinal. Disease in the colon is by far most common. Only 1% of clinical cases of amebiasis involve the liver (Haque et al., 2003), which is the primary site of extraintestinal disease. Of the intestinal forms, acute diarrhea and dysentery account for 90% of cases (Espinosa-Cantellano and Martínez-Palomo, 2000). Amebic colitis is rare and develops gradually over a period of weeks. Patients presenting with amebic colitis often have multiple discrete lesions of varying stages. Symptoms are variable, but most commonly manifest as mild to moderate abdominal discomfort/pain and frequent loose-watery stools containing variable amounts of blood and mucus (Adams and MacLeod, 1977a). Pathology ranges from superficial erosion of the colonic mucosa, to deep flask-shaped ulcers with edges that undermine the mucosa and extend deep into the muscularis and serosa. For therapy

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nitromidazoles, especially metronidazole, are given followed by an agent to clear any persisting luminal infection; rarely is surgical intervention required (Haque et al., 2003). More unusual manifestations of intestinal amebiasis are: amebomas (amebic granulomas), which are white nodular masses that protrude into the lumen and can, obstruct the bowel, toxic mega colon (recognized as a consequence of corticosteroid treatment) and fulminating amebic colitis. The latter complication is severe and onsets rapidly, resulting in necrotic lesions that extend throughout large areas of the colon, often perforating the bowel. Mortality from fulminating amebic colitis is high (Ellyson et al., 1986) and bowel resection is often required to remove necrosed segments. Extraintestinal disease may occur simultaneously with amebic colitis, but often arises in the absence of any intestinal symptoms and stool examinations are usually negative for trophozoites and cysts (Adams and MacLeod, 1977b). In fact, infections at extraintestinal sites can develop months to years after a colon infection (Stanley, 2003). Curiously, extraintestinal cases of amebiasis occur predominantly in adult males (Stanley et al., 1991). In liver, amebic infection results in formation of multiple granulomas that expand and coalesce to form large singular abscesses (Chadee and Meerovitch, 1984). In most cases symptoms develop over a 2–4 week period. They most commonly consist of dull pain in the upper-right quadrant, epigastrium, lower right chest and the right shoulder tip (Adams and MacLeod, 1977b). Less frequently patients have pain on the left side, indicating an abscess in the left lobe, which carries the risk of rupture into the pericardial sac (Adams and MacLeod, 1977b). For therapy nitromidazoles are also effective (Haque et al., 2003). In cases where patients do not respond to drugs or there is a high risk of abscess rupture, abscesses are aspirated; with large lesions up to 5000 mL of pus can be removed (Adams and MacLeod, 1977b). If rupturing occurs, it is possible for the parasites to disseminate to other organs, such as the pericardium, lungs and brain (Sullivan and Bailey, 1951; Powell and Naeme, 1960; Adams and MacLeod, 1977b; Teramoto et al., 2001) Remarkably, when treated recovery from amebic abscesses involves minimal scarring. The events marking the progression of invasive amebiasis at intestinal and extraintestinal (liver) sites are well characterized in patients and animal models at the tissue level (Fig. 1). However, our understanding of the immune mechanisms mediating the host–parasite relationship is incomplete. The purpose of this review is to present a current understanding of the host immune response during intestinal and extraintestinal amebic infection and its role in the pathogenesis of amebiasis, and to highlight areas that could shed crucial insight to the host–parasite interaction.

preventing rapid host death. A peaceful co-existence requires that the parasite has evolved ways to interact with the intestinal immune compartment that permit its survival. Given these criteria and that a harmonious relationship is usually maintained, implies that the normal host–parasite encounter leads to a tolerogenic/ hyporesponsive immune state. In this regard, secreted amebic components are known to induce a cytoprotective response in human intestinal epithelium (Kammanadiminti and Chadee, 2006), indicating that E. histolytica like other commensal microbes, has evolved to modulate signaling by intestinal epithelial cells (IEC) to direct non-inflammatory host responses. IEC are the first cells to encounter microbial/parasite antigens. Their recognition of microorganisms is critical for both initiation and regulation of microbial immune responses. Through the secretion of cytokines, chemokines and anti-microbial peptides they orchestrate innate and adaptive immunity in the gut. IEC express an array of pathogen recognition receptors (PRR), including Toll-like receptors (TLR) which recognize microbial-associated molecular patterns such as lipopolysaccharide, lipoproteins, flagellin and DNA containing unmethylated CpG. Upon binding their cognate ligand, PRR trigger activation of NFjB which in turn activates transcription of pro-inflammatory genes. In resting cells, NFjB is sequestered in the cytoplasm by IjB that hides its nuclear localization sequence. Upon phosphorylation of IjB, which signals IjB ubiquitin-mediated degradation, NFjB is released and translocates to the nucleus where it regulates transcription. Though, gut homeostasis requires continuous activation of NFjB by TLR signaling in response to intestinal bacteria (Nenci et al., 2007), commensal microbes also disrupt NFjB signaling to attenuate proinflammatory IEC responses (Artis, 2008). E. histolytica appears to be no exception. Secreted components from trophozoites induce a protective stress response in human colonic epithelial cells through an interaction with macrophages that suppresses activation of NFjB and increases resistance of the epithelium to apoptosis (Kammanadiminti and Chadee, 2006). Secreted amebic components induced expression and activation of HSF (heat shock factor)-1, a transcriptional activator of heat shock proteins (Hsp), which in turn up-regulated expression of Hsp 27 and Hsp 72. Hsp 27 then bound the catalytic subunits of IKK (IjB kinase), which phosphorylate IjB, the inhibitor of NFjB subunit p65. This attenuated translocation of p65 to the nucleus and activation of proinflammatory gene expression. Consistent with a stress response, epithelial cells treated with secreted amebic components following macrophage priming were more resistant to apoptosis. Thus, it appears E. histolytica elicits a stress response in the epithelium that promotes a hyporesponsive state towards trophozoites in the intestinal lumen.

2. Intestinal infection

2.1.2. Interleukin-10 Histological studies tracking the early events of trophozoite invasion have highlighted the importance of an intact mucosal barrier (Prathap and Gilman, 1970; Chadee and Meerovitch, 1985). In these studies, breakage in the epithelium was seen before parasiteepithelial cell contact and only after this event did invasion of mucosa and submucosa occur (Chadee and Meerovitch, 1985). Mucosal barrier integrity absolutely requires interleukin (IL)-10, a potent anti-inflammatory cytokine, and it appears to be a predetermining factor for amebic invasion (Hamano et al., 2006). IL-10 gene deficient mice have compromised and highly permeable mucosal barriers. Starting at 4 weeks of age, they develop spontaneous intestinal inflammation and extensive injury in response to normal microflora (Madsen et al., 1999). A murine amebic colitis model demonstrated that a hematopoietic source of IL-10 (likely CD4+ T cells) acting upon the non-hematopoietic compartment (likely IEC) is required for innate resistance to parasite invasion (Hamano et al., 2006). Different mouse strains are variably

2.1. Asymptomatic infections 2.1.1. Anti-inflammatory responses by intestinal epithelial cells The gastrointestinal tract is lined by a highly permeable singlelayered epithelium that interfaces with an enormous number of microorganisms. The colon is the most heavily populated region of the gastrointestinal tract, containing 109–1012 organism’s mL 1 (Artis, 2008). Thus, in addition to its absorptive role, the gastrointestinal mucosa must tightly regulate immune responses to remain vigilant against pathogens while not activating inflammatory immunity to harmless microbiota. To do so, it must precisely discriminate between commensal and pathogenic organisms. Fascinatingly, E. histolytica occupies either of these roles. Conversely, to complete its life cycle and maximize fitness, E. histolytica should establish a long-term colonic infection. Therefore, trophozoites must avoid elimination by the host immune response while also

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L. Mortimer, K. Chadee / Experimental Parasitology 126 (2010) 366–380 Ingestion of infectious cysts, excystation and migration of trophozoites to colon 1a host tolerance

Commensal in lumen

Trophozoite colonization

1b loss of tolerance tolerance not established

asymptomatic no pathology

2e Invasion of colonic mucosa

no inflammation trophozoites slip by undetected

3b trophozoites evade intravascular immunity

Trophozoites in portal circulation acute inflammatory response

2d

diarrhea/dysentery

3a intravascular immunity detect and kills trophozoites

CLEAR TROPHOZOITES TROPHOZOITES PERSIST 2a

2c

2b

adaptive Th2 response

adaptive Th1 response

disease resolved

disease resolved

4a

Liver

innate response detects and kills

disease progression no disease 4b trophozoites survive innate response, adaptive Th2 response and granulomas develop

non-healing intestinal lesions

trophozoite elimination

Event

amebic liver abscess

Host immune response

Pathology

Trophozoite colonization IL-10

• maintians intestinal homeostasis, mucosal barrier integrity Host tolerance (1a)

anti-Gal-lectin sIgA • prevents trophozoite adherence to mucus layer and epithelial cells

none

epithelium anti-inflammatory stress response • tolerence to trophozoites on apical side of epithelium Loss of tolerance (1b)

unknown • likely multifactorial involving host, parasite and possibly bacterial interactions

?

Invasion of colonic mucosa

• epithelium produces inflammatory Acute inflammatory response (1b)

cytokines: IL-1β/α, IL-8, TNF-α, GM-CSF, MCP-1

• recruitment of neutrophils, monocytes, DC, macrophages

• neutrophils and macrophages release ROS and NO into infected tissue

Th1 response (2b) • IL-12, IFN-γ, iNOS Adaptive immunity (2b,c)

mild/moderate epithelial and subepithelial inflammation diarrhea, dysentery trophozoites eliminated from tissue (2a)

disease resolved (2b)

Th2 response (2c)

• IL-4, IL-13, IL-5, Arginase-1 Th17 response (2c)

non-healing intestinal lesions (2c)

• IL-17A and IL-23, possibly recruit neutrophils during adaptive response

• role in disease not established Trophozoites in portal circulation complement • C5b-9 lyses susceptible trophozoites Intravascular immunity (2d,e,3a,b)

anti-parasite IgG • recruits complement and immune cells to trophozoites oxidative attack • intravscular neutrophils and macrophages detect and kill trophozoites

none, may be concurrent with amebic colitis trophozoites eliminated (3a) or evade (3b) intravascular immunity

Liver inflammatory response (4a,b) • iNKT cells, neutrophils, macrophages are activated and coordinate killing of trophozoites Th2 response (4b) Invade liver (4a,b)

• IL-4, IL-13 • alternatively activated macrophages • granuloma development Deactivated macrophages (4b)

• MHC II down-regulated, unresponsive to inflammatory stimuli, unable to kill trophozoites

inflammatory foci form around trophozoites; innate response detects and eliminates (4a) trophozoites Granuloma, liver abscess (4b)

Fig. 1. Schematic of host immunity and pathology during Entamoeba histolytica infection. Abbreviations: DC, dendritic cells; Gal-lectin, Galactose/N-acetyl Galactosamine inhibitable lectin; GM-CSF, granulocyte macrophage-colony stimulating factor; IFN, interferon; Ig, immunoglobulin; IL, interleukin; iNOS, inducible nitric oxide synthase; MCP, monocyte chemoattractant protein; MHC, major histocompatibility complex; (i)NKT, (invariant) natural killer T; NO, nitric oxide; ROS, reactive oxygen species; TNF, tumor necrosis factor.

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susceptible to intestinal amebic infection by intracecal inoculation of virulent trophozoites. In particular, the B6 background is completely resistant. Intriguingly, B6 immunity precludes innate recognition of the parasite as MyD88-deficient, IL-12p40-deficient, phagocyte oxidase-deficient, NO synthase-deficient and neutrophil depleted B6 mice remain resistant (Houpt et al., 2002; Asgharpour et al., 2005; Hamano et al., 2006). However, IL-10-deficient B6 mice are highly susceptible to intestinal invasion. Thus, integrity of the mucosal barrier mediated through activities of IL-10 is necessary for resistance to invasive intestinal amebiasis. IL-10 works multiple ways to promote an anti-inflammatory homeostatic environment in the gut. It dampens pro-inflammatory NFjB signaling in intestinal epithelial cells, positively effects MUC2 production, suppresses activation of antigen presenting cells, promotes induction of CD4+ regulatory T (Treg) cells, aids B cell class-switching to immunoglobulin (Ig) A and has anti-apoptotic effects on the epithelium (Ruiz et al., 2005; Schwerbrock et al., 2004; Grazia Roncarolo et al., 2001; Zhou et al., 2004). In humans, individuals that produce adequate IL-10 may develop tolerogenic/hyporesponsive states towards the parasite and therefore asymptomatic infections, while an IL-10 deficiency would impair this response. Instead, individuals lacking sufficient IL-10 would have thinned mucus layers that allow trophozoites and their secreted molecules to contact epithelial cells and underlying immune cells, which are already pre-disposed to developing inflammatory responses and abnormal regulatory responses. A lack of IL-10 could be caused by multiple factors, including genetic makeup, on-going immune responses and nutritional status. 2.1.3. Secretory immunoglobulin A Asymptomatic E. histolytica infections, like those with the closely related non-pathogenic species Entamoeba dispar, elicit intestinal IgA responses to the major parasite surface adhesin, Gal-lectin (Ravdin et al., 2003; Haque et al., 2001, 2006). In vitro, inhibition of Gal-lectin prevents trophozoite adherence to mucus, epithelial cells, and immune cells (Chadee et al., 1987; Saffer and Petri, 1991; Guerrant et al., 1981; Ravdin et al., 1988; Huston et al., 2003). Specifically, monoclonal antibodies that recognize the Gallectin carbohydrate recognition domain (CRD) inhibit trophozoite binding to colonic mucins and mammalian cells (Chadee et al., 1987; Lotter et al., 1997). Taken together, these data suggest that intestinal anti-Gal-lectin IgA would reduce trophozoite colonization in the gut. sIgA is a central component of non-inflammatory gut defenses which mediate quiescent host–microbe relationships. sIgA is produced by plasma cells in the lamina propria and is the dominant immunoglobulin at the intestinal surface. sIgA is composed of two monomeric IgA linked by the J chain and the secretory component (SC) polypeptides. Assembly takes place in lamina propria plasma cells and IEC. In plasma cells monomeric IgA is linked via the J chain to form dimeric IgA, which is then secreted and bound by the polymeric Ig receptor (pIgR) on the basolateral surface of IEC, which endocytose and transport it across the epithelium for secretion at the luminal surface (Cerutti and Rescigno, 2008; Mostov and Deitcher, 1986). Dimeric IgA joined to the SC, is then cleaved from pIgR and released into the mucus layer where it remains anchored in mucus by carbohydrate moieties present on the SC (Phalipon et al., 2002). sIgA fulfills many functions in the mucus layer; it maintains an appropriate balance of enteric microbial communities, sterically hinders microbial epitopes that mediate adherence to IEC, neutralizes inflammatory products, facilitates M (microfold) cell sampling of intestinal antigen and transports microbes that penetrate the epithelial barrier back to the lumen (Fagarasan and Honjo, 2003; Cerutti and Rescigno, 2008). Evidence shows intestinal anti-Gal-lectin IgA responses are important for human resistance to colonization and invasion. In a prospective

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study of Bangladeshi children living in an urban slum, those with stool IgA recognizing Gal-lectin CRD were protected from developing new infections (Haque et al., 2001, 2006). In South Africa, a prospective study showed robust peaks of intestinal anti-Gal-lectin IgA were associated with the ability of recovered amebic liver abscess (ALA) patients to clear the parasite and remain completely free of intestinal infection (Abd-Alla et al., 2006). An animal vaccination model showed Gal-lectin immunization elicits this response and mediates resistance to intestinal invasion, as mice with prechallenge fecal IgA that recognized Gal-lectin neared complete resistance to cecal infection (Houpt et al., 2004). Beyond preventing trophozoite adherence, E. histolytica-specific sIgA likely acts multiple ways to prevent invasion and maintain a commensal interaction between host and parasite. In addition to the roles described above, sIgA is also known to pacify host–microbe interactions by modulating metabolic pathways. An experimental system in germ-free RAG deficient mice (no T and B cells) colonized by a single commensal species of bacteria, carrying ‘hybridoma backpacks’ that produced bacteria-specific sIgA, demonstrated sIgA profoundly affects metabolism of both the host and microbe (Peterson et al., 2007). In host cells, sIgA down modulates inflammatory responses including expression of inducible nitric oxide synthase (iNOS). In bacteria, sIgA down-regulates the expression of epitopes eliciting inflammatory responses and genes involved in metabolizing toxic oxidative products. A similar situation may be true of asymptomatic E. histolytica infections. It is unclear why the parasite transitions to a pathogen. Tissue invasion requires that trophozoites have adapted to survive in an oxygenated environment. Anti-parasite sIgA could suppress inflammatory host responses which up-regulate E. histolytica metabolic pathways that neutralize oxidative species. In the absence of anti-parasite sIgA the host may elicit a more robust inflammatory response, which drives E. histolytica from a commensal to a pathogen by increasing its ability to survive in high levels of oxygen. This is speculation, however, inflammatory responses and engagement of Gal-lectin are known to alter E. histolytica metabolism. For example, superoxide produced by the oxidative burst induces trophozoites to express an iron containing superoxide dismutase that detoxifies superoxide (Bruchhaus and Tannich, 1994). Interactions between bacteria and trophozoites alter expression of E. histolytica virulence factors (Bracha and Mirelman, 1984; Wittner and Rosenbaum, 1970; Padilla-Vaca et al., 1999; Galván-Moroyoqui et al., 2008). One study found Escherichia coli that engage E. histolytica via Gal-lectin lowered parasite virulence, which was associated with reduced expression of the Gal-lectin light subunit (Padilla-Vaca et al., 1999), suggesting that engagement of the Gal-lectin could induce a more ‘commensal’ phenotype. Thus, it is plausible that engagement of parasite surface molecules by sIgA could have similar effects. Nonetheless, it is clear that anti-parasite sIgA protects the host in unperturbed conditions. Interestingly, parasite cysteine proteinases (CP) cleave human IgA (Kelsall and Ravdin, 1993; Garcia-Nieto et al., 2008). Of the two IgA subclasses, IgA2 is the dominant isotype produced in the colon due to its higher resistance to bacterial IgA proteases than IgA1 because of a 13 amino acid deletion in the hinge region of IgA2 that contains the IgA1 recognition site (Macpherson et al., 2008). However, E. histolytica CP cleave both IgA isotypes (Garcia-Nieto et al., 2008). In terms of parasite survival this may decrease the affinity of IgA for amebic antigens, thereby reducing parasite expulsion from the lumen. CP may also cleave the Fc region that interacts with surface receptors and mediates effector functions. If Fc cleavage occurred, ameba could mask immunogenic surface molecules with inert Fab fragments, providing a mechanism for the parasite to avoid detection by the immune system (see Section 2).

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2.1.4. Unexplored mechanisms of the commensal interaction Understanding how the gastrointestinal immune compartment responds to develop a commensal interaction with E. histolytica is crucial for elucidating how this interaction is subsequently lost or not established in the first place, which by extension creates an environment permissive to invasion and in some cases subversion of host immunity. With one exception (where an anti-inflammatory IEC response to E. histolytica was shown), this aspect of the host–parasite relationship has been overlooked. Induction of T regulatory cells is the usual response to harmless/commensal microbiota and in this regard several types of inducible Treg subsets have been described: inducible Foxp3+ T cells, IL-10 producing T regulatory (Tr1) cells, and TGF-b-producing T helper (Th) 3 cells (Belkaid and Oldenhove, 2008). Intestinal dendritic cells (DC) orchestrate the T cell response, and emerging evidence shows particular subsets in the lamina propria and mesenteric lymph nodes drive Treg differentiation and expression of gut-homing receptors (Coombes et al., 2007; Sun et al., 2007; Annacker et al., 2005; Johansson-Lindbom et al., 2005). Recent research has revealed that retanoic acid (RA), the biologically active metabolite of vitamin A, plays a vital role in this process; intestinal antigen presenting cells, particularly gut derived DC, are primary targets and producers of RA which require it to induce Tregs, gut-homing receptors and IgA production (Iwata et al., 2004; Mora et al., 2006; Coombes et al., 2007; Kawaguchi et al., 2007; Sun et al., 2007). Thus, a vitamin A deficiency during intestinal amebic infection would profoundly impact on an individual’s ability to mount protective tolerogenic immunity to the parasite. Not only would they have a compromised regulatory response, but impaired secretion of parasite-specific secretory IgA that can reduce trophozoite adherence (Chadee et al., 1987; Saffer and Petri, 1991; Guerrant et al., 1981; Petri et al., 1989) and subsequent overgrowth in the lumen. Vitamin A deficiency could instigate a vicious cycle, where individuals lose their ability to maintain gut homeostasis and their ability to mount appropriate anti-microbial responses once invasion ensues. Exploring the interaction between E. histolytica and intestinal APC could shed important insight on the asymptomatic amebic infection. 2.2. Invasion 2.2.1. Host recognition: E. histolytica molecules that activate inflammatory signaling Why E. histolytica invades is unknown. Human biopsy specimens and experimental animal infections show a robust inflammatory response mounted by the host in the early to intermediate stages of intestinal disease (Prathap and Gilman, 1970; Chadee and Meerovitch, 1985). Initially, the IEC barrier remains intact and trophozoite-IEC contact is not observed. However, there is moderate inflammation and neutrophil influx below the epithelium and increased lymphocyte proliferation in nearby lymphoid tissue (Prathap and Gilman, 1970). Thus, secreted molecules from the parasite that transit the epithelium must be triggering this effect. Coupling of parasite molecules to PRR initiates cellular recognition of E. histolytica. The binary signals produced by PRR signaling identify the nature of the microbial antigen and are a pre-requisite for both inflammatory and non-inflammatory responses. It is unknown how the host switches perception of E. histolytica from commensal to pathogen through PRR signaling. It is, however, known, that a variety of molecules secreted by E. histolytica engage PRR and host surface receptors that activate inflammatory pathways. 2.2.1.1. E. histolytica lipophosphopeptidogylcan. Trophozoites abundantly express lipophosphopeptidogylcan (EhLPPG) in their membranes (Maldonaldo-Bernal et al., 2005). Like Gal-lectin, EhLPPG

is both membrane bound by a GPI anchor (Lotter et al., 2009), where it plays an adhesive role, and is secreted. TLR2 and TLR4 recognize EhLPPG and activate NFjB upon ligation (Maldonaldo-Bernal et al., 2005). Recently, the complete structure of EhLPPG was elucidated. The phosphatidylinositol moiety of the GPI anchor is present in two isoforms, and one possesses two acyl chains which induces IFN-c in a TLR2/6-dependent fashion (Lotter et al., 2009). This is consistent with the structure of TLR2/6 ligands, as TLR2 and TLR6 form heterodimers that recognize di-acylated lipoproteins (Gay and Gangloff, 2007). Peritoneal monocytes and macrophages exposed to EhLPPG secrete pro-inflammatory TNF-a, IL-8 and IL-12. They also produce IL-10 and IL-6 (Maldonaldo-Bernal et al., 2005). Interestingly, high doses of EhLPPG down-regulates TLR2 gene expression (Maldonaldo-Bernal et al., 2005). Thus, EhLPPG driven signaling may activate a negative feedback loop that attenuates inflammatory responses. Effects of EhLPPG on intestinal epithelium have not been investigated. However, human IEC express low levels of TLR4 and its co-receptor MD2 that renders them hyporesponsive to TLR4 ligands (Abreu et al., 2001). Thus, EhLPPG might only mediate pro-inflammatory effects when it has permeated the intestinal barrier. Intriguingly, E. histolytica synthesizes and secretes PGE2 (Dey et al., 2003) which disrupts IEC tight junctions through binding to E-prostanoid-4 (EP4) receptors (Lejuene, unpublished results). Upon tight junction disruption, ameba components including EhLPPG, are transferred to the basolateral surface of IEC (Lauwaet et al., 2004). Breach of these molecules into the internal milieu may be the initial event that triggers early mucosal inflammation to E. histolytica. In addition to its effect on tight junction integrity, ameba PGE2 also induces IEC inflammatory responses via EP4 receptor signaling (Dey and Chadee, 2008). 2.2.1.2. Gal-lectin. After tight junctions are disturbed, Gal-lectin is also transferred across epithelial layers (Leroy et al., 1995; Lauwaet et al., 2004). Although PRR interacting with Gal-lectin have not been identified, its pro-inflammatory effects on several cell types are known. In naive/unconditioned macrophages and DC, Gal-lectin activates NFjB and Mitogen-activated protein kinases (MAPK) (Kammanadiminti et al., 2003; Ivory and Chadee, 2007). In macrophages, the Gal-lectin CRD up-regulated expression of IL-1b, IL-1a, TNF-a and a number of genes involved in NFjB and MAPK signaling (Séguin et al., 1995; Kammanadiminti et al., 2003). In contrast to LPPG, TLR2 gene expression was up-regulated by the CRD while TLR4 expression was suppressed (Kammanadiminti et al., 2003). The implications of this are not clear. However, signals received through different PRR are integrated to produce a tailored immune response that dictates the type of adaptive immunity generated. Thus, altered TLR expression could influence the adaptive immune response to E. histolytica. In DC Gal-lectin stimulated IL-12 production, a key Th1 inducing cytokine, and induced DC to drive naive CD4+ T cells into effectors with a Th1 phenotype (Ivory and Chadee, 2007). 2.2.1.3. E. histolytica DNA. Intriguingly, E. histolytica DNA (EhDNA) is a TLR9 ligand (Ivory et al., 2008). TLR9 recognizes unmethylated CpG dinucleotides and activates signaling in acidified intracellular compartments that leads to robust pro-inflammatory responses and induction of Th1 immunity (Wagner, 2002). In humans CpG sequences are usually methylated at position 5 of the cytosine ring, whereas in microbial DNA these sites are unmethylated. As the E. histolytica genome is A:T rich (Ivory et al., 2008), it was unexpected that EhDNA activates TLR9. In early stages of invasive infections, TLR9–EhDNA engagement could overcome tolerogenic mechanisms towards the parasite, as TLR9 activation on APC and Tregs is known to limit Treg suppressor functions (Larosa et al., 2007; Pasare and Medzhitov, 2003). Trophozoite destruction by early effector cells (neutrophils) may release EhDNA that activates

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TLR9 signaling. Subsequently, this could aid development of a protective Th1 immune response that allows the host to clear an invasive infection. 2.2.1.4. Cysteine proteinase 5. Studies in our laboratory have recently demonstrated that E. histolytica cysteine proteinase 5 (EhCP5) instigates IEC inflammatory responses in a non-proteolytic fashion through integrin engagement, and for the first time a pathogenic role for the pro-enzyme form of EhCP5 (Hou et al., unpublished results). EhCP5 is one of the most abundantly expressed CP and is exclusively expressed by E. histolytica, and not by the closely related non-pathogenic species E. dispar (Bruchhaus et al., 2003). EhCP5 is both membrane bound and secreted (Jacobs et al., 1998), and thus far only proteolytic functions of EhCP5 have been ascribed roles in pathogenesis. E. histolytica CP are synthesized as pro-enzymes that are cleaved to mature forms, and both pro- and mature-EhCP5 are secreted (Hou et al., unpublished results). We have recently shown (Hou et al., unpublished results) that both pro- and mature-EhCP5 have an RGD motif that engages integrin avb3 and triggers PI3 kinase phosphorylation of integrin linked kinase (ILK). In turn, phosphorylated ILK activates the Akt pathway and drives NFjB pro-inflammatory gene expression both in vitro and in vivo. Thus, in addition to its proteinase activity EhCP5 interacts with integrins in a fashion analogous to PRR. EhCP112 and EhCP118 also contain RGD motifs (Bruchhaus et al., 2003), but these enzymes are not highly expressed or secreted by ameba. 2.2.2. Acute mucosal inflammatory response 2.2.2.1. Inflammatory response in epithelial and subepithelial cells. Inflammation is a double-edged sword. On one hand it protects the host from invasive pathogens, while on the other it can lead to severe tissue damage. In the context of invasive intestinal infections, the initial inflammatory response to E. histolytica is believed to aggravate and possibly drive pathogenesis. Histological studies indicate that trophozoites enter the lamina propria through micro-erosions in the epithelia (Prathap and Gilman, 1970; Chadee and Meerovitch, 1985), which are possibly caused by inflammation below the epithelium (Chadee and Meerovitch, 1985). Thus, the parasite may enter host tissue passively, directly facilitated by the host inflammatory response. In vivo models corroborate this theory. In a SCID mouse–human intestinal xenograft model, infection of the xenograft with trophozoites elicits a robust inflammatory response from the grafted tissue, characterized by strong IL1b and IL-8 expression, an early neutrophil influx and extensive damage to the intestinal graft (Seydel et al., 1997a). When NFjB driven gene expression is blocked, damage to intestinal xenografts is prevented, intestinal barrier integrity is maintained and neutrophil influx is attenuated (Seydel et al., 1998). IL-1b and IL-1a are key mediators of cytokine induced proinflammatory responses, signaling through IL-1RI (a member of IL-1R/TLR superfamily) via the same intracellular pathways used by TLR. They activate transcription of genes involved in acute inflammatory responses such as IL-8, which is a potent neutrophil chemoattractant and activator. In vitro, human cell lines respond to trophozoites similar to the in vivo response, by releasing IL-1a and IL-1b and by producing an array of neutrophil chemoattractants including IL-8, IL-6 and GM-CSF (Eckmann et al., 1995). Although, intestinal cells lines mount a dampened response to E. histolytica compared to cells of extraintestinal tissue origin (Eckmann et al., 1995), likely because intestinal epithelium which continuously contacts microorganisms is engineered to be more hyporesponsive. However, E. histolytica CP can enhance early mucosal inflammation by cleaving released pre-IL-1b into its active form, thereby triggering a cytokine-induced inflammatory response (Zhang et al., 2000). In vivo, EhCP5 has been shown critical for pathogenesis, as ameba

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that are deficient in EhCP5 fail to induce intestinal inflammation, owing to both the proteolytic and non-proteolytic functions of EhCP5 which activate NFjB through cleavage of pre-IL-1b and integrin binding (Hou et al., unpublished results), respectively. 2.2.2.2. Acute neutrophilic response: protective or destructive?. The current opinion is that the epithelium and possibly underlying immune cells release inflammatory signals that recruit an acute neutrophilic response, and that neutrophils are largely responsible for exacerbating intestinal injury. In vitro, adding neutrophils to intestinal monolayers that are challenged with trophozoites enhances epithelial cell destruction (Burchard et al., 1993). In vivo, inhibiting pro-inflammatory gene expression in intestinal xenografts in response to virulent trophozoites prevents neutrophil recruitment, corresponding to a lack of intestinal destruction (Seydel et al., 1998). Together, these data suggest neutrophils play a detrimental role in the disease process, by possibly inflicting mucosal damage that facilitates parasite entry into the lamina propria. However, there is evidence suggesting neutrophils are also protective. In a murine amebic colitis model, neutrophil depletion with a Gr-1 monoclonal antibody (Gr-1 is a marker of peripheral neutrophils) from partially resistant strains dramatically increased susceptibility to and severity of disease (Asgharpour et al., 2005). Challenged mice lacked intestinal myeloperoxidase activity (a hallmark of neutrophil activity) and exhibited severe cecal inflammation. Interestingly, SCID mice (no T and B cells) on partially susceptible backgrounds were less susceptible to amebic infection and exhibited no difference in pathology at 30 days post-infection, suggesting that innate immune cells are crucial for clearing invasive parasites (Asgharpour et al., 2005). Thus, neutrophils have two opposing -both protective and damaging- roles in intestinal disease pathogenesis. The primary function of neutrophils is to isolate, kill and engulf pathogens. Neutrophils have an array of anti-microbial defenses that makes them highly equipped for killing. They express surface receptors that aid phagocytosis, including CR3 (complement receptor 3) and FccR that binds complement C3b and IgG on surfaces of opsonized pathogens. These enable neutrophils to internalize pathogens into phagosomes, which fuse with lysosomes containing NADPH oxidase and myeloperoxidase that generate toxic oxygen species. They have azurophilic granules containing myeloperoxidase, cathepsins, azurocidin, elastase, proteinase 3 and defensins, and release these into infected tissue. They thrust out NETs (neutrophil extracellular traps) that are fibrous networks of chromatin covered in proteases that can trap invading microorganisms (reviewed in Hickey and Kubes, 2009). Most important for E. histolytica, they extracellularly generate reactive oxygen species (ROS) using NADPH oxidase located in the cell membrane; early studies showed ROS kill trophozoites and that highly virulent strains are less susceptible to ROS (Murray et al., 1981; Ghadirian et al., 1986). The neutrophilic response in amebic infections represents a catch-22; their degranulation elevates tissue destruction (Salata and Ravdin, 1986) and in vivo most certainly contributes to pathology, while their production of H2O2 is protective and does not contribute to tissue damage, as catalase (H2O2 scavenger) or neutrophils from patients with granulomatous disease (incapable of producing an oxidative burst) in tissue cultures with trophozoites do not reduce neutrophil damage (Salata and Ravdin, 1986). Host cell integrity is likely compromised by proteases released from neutrophil granules. Whether neutrophils tip the balance towards parasite clearance or the progression of disease may depend on both host and parasite factors. On the host side, proper neutrophil activation is required, as in vitro interferon (IFN)-c and tumor necrosis factor (TNF)-a stimulated neutrophils kill ameba via contact-dependent and contact-independent manners (Denis and Chadee, 1989). However,

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amebae are also highly capable of disarming and inflicting neutrophil destruction. In vitro, highly virulent trophozoites can kill human neutrophils at a ratio of one trophozoite to 3000 neutrophils, while even low virulent strains can accomplish this at a 1:400 ratio of trophozoites to neutrophils (Guerrant et al., 1981). For defense, E. histolytica expresses several enzymes that dismantle the oxidative burst, including a superoxide dismutase (Bruchhaus and Tannich, 1994, 1995) and a bifunctional NADPH: flavin oxidoreductase (Bruchhaus et al., 1998) that converts superoxide anions to H2O2 and a surface perioxiredoxin that detoxifies H2O2 (Choi et al., 2005; Davis et al., 2006). The latter enzyme appears critically important for protecting E. histolytica against oxidative killing, as H2O2 is highly damaging to trophozoites (Murray et al., 1981; Ghadirian et al., 1986). Furthermore, the perioxiredoxin is expressed 50-fold higher in E. histolytica than E. dipsar, which is highly susceptible to H2O2 (Choi et al., 2005). Also, E. histolytica perioxiredoxin is expressed predominantly in the cell membrane and localizes to the point of contact with host cells, whereas E. dispar perioxiredoxin is expressed intracellularly. The parasite also secretes a serine protease inhibitor, Ehserp, which binds and inactivates cathepsin G released by degranulating neutrophils (Riahi et al., 2004). After neutralizing neutrophil defenses, trophozoites are able to destruct them via engagement of b2 integrins that leads to activation of NADPH oxidase and ROS-mediated apoptosis (Sim et al., 2007). Trophozoites rapidly follow this by phagocytosing neutrophil corpses (Marion and Guillén, 2006; Velazquez et al., 1998), which reduce intestinal inflammation and might facilitate parasite evasion of host immunity. 2.2.2.3. Roles of other innate immune cells. As most individuals resolve invasive intestinal amebiasis after an acute inflammatory response, corresponding with an episode of inflammatory/secretory diarrhea and dysentery, it is plausible that in most cases a neutrophilic response is protective. However, there is a paucity of information regarding the contributions made by other types of innate immune cells in this early phase of host defense. For example, unconventional T cells residing in the gut serve as sentinels of innate immunity and may enable immediate host responses to invading trophozoites. Recently, iNKT activation was shown critically important for protecting against amebic liver abscess formation (see Section 3) (Lotter et al., 2009). However, the role of iNKT cells in intestinal invasion is untested. cd T cells, whose T cell receptors (TCR) appear to recognize intact unprocessed self-antigen (Rock et al., 1994; Meresse and Cerf-Bensussan, 2009), make up 40% of colonic intraepithelial lymphocytes (Meresse and CerfBensussan, 2009). Many of these cells have effector phenotypes and the ability to immediately respond to self-derived danger signals that would be produced during E. histolytica invasion. Another innate cell of interest is the NK cell. It was recently shown that the intestine harbors distinct populations of NK cells and compared to NK cells of other peripheral sites, intestinal NK cells have greatly reduced expression of effector molecules required for cytotoxic function (Satoh-Takayama et al., 2008). A large proportion of intestinal NK cells are incapable of expressing IFN-c and TNF-a and instead express the transcription factor, RORct (retinoic acid receptor related orphan receptor gamma t) and IL-22 in response to local environmental signals. IL-22 producing NK cells were shown to be critical for immune defense against murine enteric pathogen, Citrobacter rodentium, which causes acute colitis in mice (Satoh-Takayama et al., 2008). IL-22 is required in early host defense to C. rodentium to maintain epithelial integrity and induce secretion of anti-microbial peptides (Zheng et al., 2008). As NK cells are situated in association with the epithelium and in the lamina propria, they would be poised for immediate action against invading trophozoites. A functional role for IL-22 in intestinal amebic infection has not been ascribed. It may be that intestinal NK

cells function in a non-inflammatory capacity to return/maintain barrier function during trophozoite invasion. How unconventional T cells and NK cells influence gut immunity is largely still being elucidated. Our knowledge of these cells in intestinal amebiasis in asymptomatic or pathological conditions is completely unknown. 2.2.3. Amebic ulcers and non-protective immune responses Although most intestinal invasions are cleared after an acute inflammatory response, E. histolytica escapes elimination in a small percentage of individuals and intestinal lesions develop. Curiously, this phase is marked by an absence of severe inflammation (Prathap and Gilman, 1970). This more chronic state is associated with development of a non-protective adaptive immune response. Before trophozoites move into the subepithelial zone, small focal erosions in the mucosa occur. The lamina propria is infiltrated with not only neutrophils, but also macrophages, plasma cells, lymphocytes and eosinophils and there is continued proliferation within nearby lymphoid follicles (Prathap and Gilman, 1970). Trophozoites become abundant at the sites of epithelial disruption and are absorbed in a thick purulent exudate. Next they are seen below the epithelium although, tissue inflammation and destruction at this point are minimal. As pathology progresses, large numbers of trophozoites together with neutrophils create a zone of necrosis in the lamina propria. In advanced lesions deep flaskshaped ulcers form, where the edges undermine the mucosa and extend into the muscular and serosal layers. The floors of ulcers are covered in a thick exudate and are separated from viable underlying tissue by a fibrous zone that is infiltrated with eosinophils. Trophozoites are located in deep layers of ulcers and in some instances in undamaged tissue nearby. At this point immune cells are absent inside the lesions, but surrounding tissue has neutrophil and plasma cell infiltrates. It is also noted that advanced lesions have relatively mild inflammation compared to the extent of tissue destruction (Prathap and Gilman, 1970). At this stage, adaptive immune responses are critical for resolving or perpetuating invasive intestinal infections. Human data and in vitro and in vivo models support a paradigm that Th1 responses in the gut clear E. histolytica, while Th2 responses are anti-protective, likely through suppressing IFN-c which is the primary Th1 cytokine produced by CD4+ T cells that can activate effector cells capable of clearing the parasite (Denis and Chadee, 1989; Lin and Chadee, 1992). A large-scale study of children in Bangladesh showed that high parasite-specific IFN-c production by blood mononuclear cells correlated with a lower risk of developing future amebic dysentery and diarrhea (Haque et al., 2007). In vitro, IFN-c activates neutrophils (Denis and Chadee, 1989) and macrophages (Lin and Chadee, 1992) to become amebicidal. In addition to oxidative burst, IFN-c stimulated macrophages express iNOS and synthesize large amounts of nitric oxide (NO), which plays a critical role in killing trophozoites (Lin and Chadee, 1992) and inhibiting parasite CP enzyme activity, which if left unchecked inflicts extensive tissue damage (Siman-Tov and Ankri, 2003). In vivo, NO plays an essential role in host protection, as mice genetically deficient for iNOS are highly susceptible to experimentally induced amebic liver abscess (see Section 3) (Seydel et al., 2000). A murine amebic colitis model revealed that adaptive Th2 responses through production of IL-4 by CD4+ cells is detrimental to the host in established gut infections (Guo et al., 2008). Susceptible mice that are cecally infected with trophozoites develop chronic non-healing lesions and produce high amounts of Th2 cytokines, including IL-4, IL-13 and IL-5, and have Arginase-1:iNOS ratios skewed heavily towards Arginase-1 (Guo et al., 2008). The Arginase-1:iNOS ratio indicates whether macrophages are classically or alternatively activated, where classically activated macrophages express iNOS and exhibit a pro-inflammatory Th1

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phenotype and alternatively macrophages express Arginase-1, are anti-inflammatory and promote wound repair processes (Gordon, 2003). Thus, macrophages with an anti-inflammatory phenotype predominate in established intestinal disease. Conversely, infected mice have suppressed expression of Th1 cytokines, IFN-c and IL12. However, depletion of either CD4+ cells (Houpt et al., 2002) or IL-4 (Guo et al., 2008) allows a Th1 response (i.e. IFN-c production) to develop and concomitantly ameliorates disease. If, however, IFN-c and IL-4 are depleted together or IFN-c is depleted alone, disease goes unresolved, demonstrating unequivocally that IFN-c plays a protective role in intestinal amebiasis. Interestingly, cecally infected mice also develop robust IL-17A expression and moderately increased IL-23 expression (Guo et al., 2008), which is characteristic though not conclusive of a Th17 response, as Th17 cells are not the only source of IL-17. IL-17A belongs to a family of six cytokines, called the IL-17 family, known for their orchestration of innate immune cells (Bettelli et al., 2007). IL-17A binds with high affinity to the ubiquitously expressed IL-17RA and induces cytokines that recruit and activate neutrophils including IL-8, GRO-a, IL-6, GM-CSF, G-CSF and CXCL-10 (Korn et al., 2009). Neutrophils are present not only in the acute stage of intestinal amebiasis but also throughout chronic stages. Thus, IL-17A production is consistent with the immunopathology of human biopsies. Th17 cells represent a heterogeneous population producing an array of cytokines, including IL-21, IL22, IL-6, IL-10 and TNF-a (Maloy and Kullberg, 2008). The functional significance of IL-17A and Th17 cells in intestinal amebiasis remains to be clarified. The current concept is that APC secrete IL-6 and TGF-b that synergize to differentiate naive CD4+ T cells into Th17 cells through expression of RORct. Differentiating T cells then become responsive to IL-23 after up-regulating their IL-23 receptor, and IL-23 contributes to Th17 cell expansion and maintenance (Korn et al., 2009). The IL-23/17 axis appears to be important for neutrophil recruitment and inhibiting development of Th1 responses (Lemosa et al., 2009). Interestingly, PGE2 can play an important role in this process through an amplification circuit that promotes IL-23 expression in DC, which in turn up-regulates COX2 and thus PGE2 synthesis, and by impairing IL-12 and IFN-c, which negatively regulate IL-17 expression (Lemosa et al., 2009; Boniface et al., 2009; Yao et al., 2009). As E. histolytica secretes PGE2, a parasite source of PGE2 could possibly condition DC to drive IL-23-induced neutrophil migration through a Th17 response and could simultaneously inhibit a Th1 response needed for clearing the infection. The conditions/signals that recruit an anti-protective Th2 response instead of a protective Th1 response towards E. histolytica are currently unknown. Initially a robust mucosal inflammatory response recruits large numbers of neutrophils as well as monocytes, immature DC and several other immune cell types to the site of infection. In this regard, trophozoites secretions also induce IEC to express monocyte chemoattractant protein (MCP)-1 through PI3 kinase signaling to Akt for NFjB activation (Kammanadiminti et al., 2007). Among others, MCP-1 is a potent chemoattractant for monocytes, immature DC and basophils (Charo and Ransohoff, 2006) and is therefore, important for recruiting unconditioned/naive APC to infected tissue. In some individuals, APC must then receive signals that are translated into Th2 immunity. There is human evidence suggesting that genetics plays a role in this response. In a prospective study of E. histolytica infection in Bangladeshi children (Haque et al., 2002), children were at increased risk for developing amebic dysentery/ diarrhea if they produced serum anti-parasite IgG not recognizing the Gal-lectin CRD. Intriguingly, these children produced parasite-specific IgG4 antibodies, which in humans are induced by Th2 cytokine IL-4. Furthermore, not all children mounted antiparasite IgG responses, instead this trait clustered within fami-

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lies (Haque et al., 2002). MHC restriction might be a pre-disposing genetic factor that biases the host towards an E. histoltyica-induced Th2 response, as an individual’s repertoire of MHC alleles determines the peptides that can be presented to T cells and therefore, dictates the type of adaptive immune response generated. In support of this possibility, the MHC class II allele DQBl*0601 is associated with resistance to E. histolytica (Duggal et al., 2004). Interestingly, the N-terminal cysteine poor region of Gal-lectin elicits anti-protective immunity in an animal model of ALA, and in humans 100% of individuals with symptomatic E. histolytica infections were found to produce serum antibodies to the cysteine poor region, whereas not all individuals with asymptomatic infections produced antibodies against this region (Lotter et al., 1997). Perhaps more important, the highest degree of protection against animal ALA is elicited by a portion of the Gal-lectin CRD recognized by only 10% of symptomatic individuals, whereas 78% of asymptomatic individuals developed antibodies against this region (Lotter et al., 1997). It has not been determined whether MHC restriction plays a role in this phenomenon.

3. Extraintestinal disease 3.1. Dissemination through the blood: immune evasion Extraintestinal dissemination requires that trophozoites are able to survive within the vasculature, which in addition to high oxygen concentration (E. histolytica lives in an anaerobic habitat and is normally highly susceptible to oxygen), is protected by a network of immune cells and humoral factors. To persist in these environment trophozoites must subvert detection by antibody and complement and continue to resist oxidative attack. Thus, mechanisms of immune evasion are critical for E. histolytica to establish extraintestinal infections.

3.1.1. Degradation of IgG In addition to degrading IgA, trophozoite CP can also inactivate circulating IgG (Tran et al., 1998). Likely, there are many consequences to this. For example, IgG cleavage could decrease the affinity of antibody for antigen, or if the Fc portion was removed, trophozoites could be disguised from the immune system by cloaking their immunogenic surface molecules in Fab fragments (Que and Reed, 2000). This would prevent activation of complement by the classical pathway and attacks from immune cells bearing corresponding Fc receptors. The parasite could then transit through the internal milieu ignored by the host immune system.

3.1.2. Capping and surface receptor cleavage Another mechanism trophozoites use to avoid targeted immune responses is surface receptor capping, which disposes surface receptors from the parasite that have been recognized by molecules of the immune system (such as Ig or complement components) (Calderón et al., 1980; Baxt et al., 2008). In this process parasite receptors are rapidly removed to the posterior end and are released in membrane bound vesicles. Recently, an active intramembrane serine protease, EhROM1, was found to redeploy to the base of cap complexes late in the capping process, and is thought to function in formation or release of the cap (Baxt et al., 2008). In addition, EhROM1 cleaves the transmembrane domain of the Gal-lectin heavy subunit (Hgl). As Hgl is the highly immunogenic component of Gal-lectin, EhROM1 could aid host evasion by continuously cleaving Hgl from trophozoite surfaces, thereby preventing Ig from recruiting immune targeting mechanisms towards the parasite.

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3.1.3. Resistance to complement Complement is a critical part of the intravascular immune system that can mediate trophozoite destruction. It is therefore necessary for trophozoites that have entered the vascular system to resist this defense. Complement is activated when zymogens in serum are cleaved and produce an enzyme cascade that results in non-specific binding of complement components to pathogen surfaces. Its two primary functions are to directly lyse foreign cells by the membrane attack complex C5b-9 and to opsonize pathogens with complement molecules that facilitate pathogen recognition by immune cells bearing cognate receptors. A recent study found sera from female adults not previously exposed to E. histolytica is profoundly more effective at killing trophozoites than sera from previously unexposed adult males through a complement-dependent mechanism (Snow et al., 2008). The prevalence of ALA is five to seven times higher in men than women, even though rates of male and female gut colonization do not appear to differ (Stanley et al., 1991). Therefore, gender differences involving some aspect of the complement system appears to be a reason why extraintestinal amebiasis predominates in males. In vitro, both E. histolytica and E. dispar activate complement by the alternative and classical/mannose binding lectin pathways (Reed et al., 1986). Interestingly, parasite CP activate the alternative pathway by cleaving C3 into functional C3b (Reed et al., 1989). The membrane attack complex C5b-9 effectively lyses susceptible trophozoites (Reed and Gigli, 1990). However, E. histolytica recovered from colitis or liver abscess patients are resistant to complement-mediated killing (Reed et al., 1986) and in vitro this resistance can be induced by culturing E. histolytica in media containing serum (Hamelmann et al., 1993). Several mechanisms contribute to the parasite’s complement insensitively. Both Gal-lectin and a recently identified 21 kDa surface protein have epitopes of the complement inhibitory protein, CD59 (Braga et al., 1992; Ventura-Juárez et al., 2009). In humans CD59 is present on cell surfaces to prevent ‘self’ lysis by inhibiting the formation of the membrane attack complex. Similarly, Gal-lectin prevents membrane attack complexes from forming on trophozoite surfaces (Braga et al., 1992). In contrast to susceptible isolates of E. histolytica, isolates that are complement resistant are not lysed by C5b-9 (Reed and Gigli, 1990), indicating perhaps that differential expression of Gal-lectin or the 21 kDa surface protein confers the parasite’s resistance/susceptibility to complement attack. Beyond this, lipophosphogylcan (LPG) and LPPG on the cell surface may also afford trophozoites protection by creating an impenetrable layer to complement components. In support, complement susceptible E. dispar has a much thinner LPG/LPPG glycolax (Bhattacharya et al., 2000). Additional evidence that Gal-lectin and LPG/LPPG are critical for evasion of complement comes from inhibiting E. histolytica GPI anchor synthesis, which localizes Gal-lectin and LPG/LPPG to trophozoite membranes. When GPI anchor formation is disrupted trophozoites become highly susceptible to complementmediated killing and unable to mount liver infections (Weber et al., 2008). At another level, E. histolytica survives complement by attenuating the effects of anaphylatoxins C5a and C3a by cleaving and inactivating them with CP (Reed et al., 1995). C5a and C3a are potent activators of inflammation; they trigger release of histamine from mast cells, lysosomal enzymes from leukocytes and pro-inflammatory cytokines including IL-6 and TNFa (Walport, 2001; Köhl, 2001; Gasque, 2004). They also increase vascular permeability and attract neutrophils, monocytes, eosinophils, mast cells, basophils, activated B cells and T cells to sites of activation (Köhl, 2001; Gasque, 2004). Anaphylatoxin blockade detracts from immune detection in the blood and reduces inflammation in amebic lesions (partially explaining the lack of severe inflammation in advanced liver and intestinal lesions).

In addition to humoral components of the intravascular immune network, several types of immune cells are present in the vasculature to detect and respond to pathogens, including neutrophils, monocytes, iNKT cells and even endothelial cells. Lymphocytes crawl and roll along the endothelium surveying contents of the blood and recruit responses when pathogens are recognized (Hickey and Kubes, 2009). Given that extraintestinal disease accounts for only 1% of clinical amebiasis cases, the cellular and humoral arms of the intravascular immune system are likely highly effective at removing trophozoites from the blood. On the other hand, trophozoites that persist are specialized to survive this microenvironment. Inductive mechanisms appear to play a role in their adaptation, however, recent studies reveal that parasite genotype is also important (Ali et al., 2007, 2008). It was found that individuals with amebic liver abscesses and concurrent colon infections harbor distinct parasite genotypes in the two locations (Ali et al., 2008). Preceding work has also alluded to this (Ali et al., 2007; Ayeh-Kumi et al., 2001), although isolates used in these studies were from separate clinical groups (asymptomatic, diarrhea/dysentery and liver abscess) that limited genotyping comparisons. Now that parasite genotyping has been done in different locations of single individuals, these data indicate that only a minor genotype reaches the liver or that DNA reorganization/ recombination events take place when trophozoites migrate to the liver (Ali et al., 2008). Although many parasite virulence factors have been identified, we still do not have a clear picture of which ones determine the outcome of infection. This aspect is also crucial for understanding the genesis of extraintestinal disease (Table 1). 3.2. Development of liver abscesses 3.2.1. Emigration from vascular system into hepatic tissue After entering and surviving in the hepatic portal system, trophozoites that establish liver abscesses must emigrate from the blood-carrying spaces in the liver, called sinusoids, into the parenchymal tissue. Little is known about the migration process used by trophozoites. The parasite might utilize surface bound EhCP5 (Hou, unpublished results) and b1EhFNR (a b1-integrin-like molecule) (Talamás-Rohana et al., 1998) to interact with integrins via the RGD motif to tether and propel along the endothelium, much like immune cells crawl along the vasculature to survey blood contents for pathogens. In addition, the sinusoidal endothelium is fenestrated (having large holes), which allows plasma in the sinusoids to enter the space of Disse (the region between sinusoids and hepatocytes), and may facilitate trophozoite migration into liver parenchyma. 3.2.2. Early inflammatory response A number of immune cells are resident in the liver including Kupffer cells that extend processes into multiple sinusoids, DC, T cells, NKT cells and NK cells (Crispe, 2009). Like the intestinal immune compartment, the liver requires a tolerogenic environment because it comes in contact with enormous amounts of food antigen and microbial products arriving from the intestine (Crispe, 2009). Thus, the same stipulations apply to the liver; it must clearly discriminate between harmless and pathogenic material. A diverse array of PRR on liver cells converge on NFjB and under the right conditions recruit inflammatory responses. Several rodent models are currently used to study ALA, and because ALA is not caught at early stages in humans, these models found our knowledge of ALA pathogenesis. Within hours of introducing trophozoites to the liver numerous inflammatory foci form and incur massive trophozoite death. However, after this initial response the few surviving trophozoites multiply rapidly and inflammatory foci quickly expand (Rigothier et al., 2002).

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Table 1 The major E. histolytica molecules involved in activating and/or evading host defense mechanisms. Abbreviations: ALA, amebic liver abscess; ECM, extracellular matrix; EhCP5, Entamoeba histolytica cysteine proteinase 5; EhLPPG, Entamoeba histolytica lipophosphopeptidoglycan; EhLPG, Entamoeba histolytica lipopeptidogylcan; EhROM1, Entamoeba histolytica rhomboid protease 1; EhSERP, Entamoeba histolytica serine protease; H2O2, hydrogen peroxide; KERP1, lysine and glutamic acid-rich protein; MUC2, (secretory) mucin 2; PGE2, prostaglandin E-prostanoid 2; PRR, pathogen recognition receptors; TLR, toll-like receptor. Host defense mechanism

E. histolytica molecules involved in evasion or activation of host defense

Mucus layer

Gal-lectin  Adherence to mucin oligosaccharides (Chadee et al., 1987) Cysteine proteinases  Cleave MUC2, dissolve mucus layer (Lidell et al., 2006)  Cleave IgA2 (Kelsall and Ravdin, 1993; Garcia-Nieto et al., 2008) Glycosidases  Cleave sugar residues attached to mucin that expose the mucin backbone to proteolytic degradation (Moncada et al., 2005)

Inflammation in intestinal epithelium and subepithelium

EhLPPG, Gal-lectin, EhDNA, PGE2, EhCP5  Engage PRR and activate inflammatory signaling pathways (Maldonaldo-Bernal et al., 2005; Kammanadiminti et al., 2003; Ivory and Chadee, 2007; Dey and Chadee, 2008) Cysteine proteinases  Degrade ECM (Que and Reed, 2000)  Cleave IgA and IgG (Kelsall and Ravdin, 1993; Garcia-Nieto et al., 2008; Tran et al., 1998) Gal-lectin  Cell adherence, apoptosis signaling, phagocytosis (Petri et al., 1987; Saffer and Petri, 1991; Huston et al., 2003)

Neutrophil attack

Superoxide dismutase, bifunctional NADPH: flavin oxidoreductase  Detoxify superoxide to H2O2 (Bruchhaus and Tannich, 1994, 1995; Bruchhaus et al., 1998) Perioxiredoxin  Localizes to site of host cell attachment and detoxifies H2O2 (Choi et al., 2005; Davis et al., 2006) Ehserp  Inactivates cathepsin G from degranulating neutrophils (Riahi et al., 2004) Engage b2 integrin  Induce ROS dependent pathway of apoptosis in neutrophils (Sim et al., 2007) EhLPPG  Binds TLR 2/6 and TLR4; activates a pro-inflammatory macrophage response (Maldonaldo-Bernal et al., 2005) Gal-lectin  CRD activates a pro-inflammatory macrophage response (Séguin et al., 1995; Kammanadiminti et al., 2003) Eharginase-1  Depletes arginine from the local environment required by macrophages to make NO (Elnkave et al., 2003) Macrophage deactivation  Molecules secreted by trophozoites locally suppress macrophages function (Denis and Chadee, 1988; Wang et al., 1994; Wang and Chadee, 1995)  Involves a PGE2-dependent mechanism; a parasite source of PGE2 may be important (Wang et al., 1994; Wang and Chadee, 1995)

Macrophage attack

NKT cell

EhLPPG  Presentation on CD1 to NKT cell TCRs in combination with TLR signaling activates NKT cells to produce IFN-c (Lotter et al., 2009)  iNKT cells are crucial for ALA protection (Lotter et al., 2006, 2009)

Complement evasion

Cysteine proteinases  Cleave IgG (Tran et al., 1998), prevents activation of classical complement pathway on trophozoite surface  Cleave C3 to activate alternative pathway of complement (Reed et al., 1989)  Cleave and inactivate anaphylatoxins C3a and C5a (Reed et al., 1995) Gal-lectin  Contains epitopes of CD59 that prevent formation of membrane attack complex; a 21 kDa protein on trophozoite surfaces may have the same function (Braga et al., 1992; Ventura-Juárez et al., 2009) EhLPPG, EhLPG  Cover trophozoite surface and are suggested to protect from complement attack (Bhattacharya et al., 2000; Weber et al., 2008) EhROM1  Proposed to remove surface molecules recognized by humoral immune components to avoid classical activation of complement and immune cells with Fc receptors for Ig  Cleaves Hgl, may remove Hgl bound by Ig (Baxt et al., 2008)  Late in surface capping, localizes to base of cap where it is proposed to function in cap formation or release (Baxt et al., 2008)

Liver

Amebapores  Pore-forming peptides, insert in cell membranes and cause necrosis and possibly apoptosis of host cells  Play major role in abscess development (Zhang et al., 2004) EhCP5  Plays a major role in abscess development (Tillack et al., 2006) KERP1  A lysine and glutamic acid-rich protein up-regulated in ALA, involved in host cell contact, required by trophozoites to form abscesses (Santi-Rocca et al., 2008)

3.2.2.1. iNKT cells. The rapid response in the liver is in part due to iNKT cells, which critically protect this tissue from E. histolytica (Lotter et al., 2006). iNKT cells seem to play a central role in coordinating and inflicting the massive trophozoite destruction that occurs within the first few hours of their arrival. iNKT cells make up a high frequency (30%) of lymphocytes in the liver in both human and mouse, which includes the Va24–Ja18 subset in humans (orthologous to Va14–Ja18 in mice) that responds to glycolipids

presented on CD1d (Crispe, 2009). Upon ligation of their TCR they can rapidly produce a variety of cytokines, including pro-inflammatory IFN-c and anti-inflammatory IL-4, IL-10 and IL-13. These have both immediate and downstream effects for initiating inflammation and directing adaptive immunity, and can critically regulate clearance or persistence of E. histolytica. Mice deficient for the Ja18 chain lack iNKT cells and develop larger abscesses than control animals after trophozoites are intrahepatically delivered

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(Lotter et al., 2006), demonstrating a protective role for iNKT cells in vivo. It was recently demonstrated that presentation of E. histolytica glycolipids on CD1d along with TLR activation stimulates iNKT cells to produce IFN-c (Lotter et al., 2009). Specifically, EhLPPG stimulated a protective IFN-c response and did not elicit Th2inducing IL-4 from iNKT cells, and protected mice from ALA. iNKT cell activation by EhLPPG was triggered in a combined TLR and CD1d dependent manner (Lotter et al., 2009). EhLPPG is a known ligand for TLR2 and TLR4, and now in the context of CD1d is recognized as a ligand for the TCRs of iNKT cells. 3.2.2.2. Neutrophilic response. Within hours of detecting trophozoites in the liver, an acute inflammatory response recruits massive numbers of neutrophils, which can be seen surrounding trophozoites (Rigothier et al., 2002). A neutrophilic liver response presents the same dichotomy as intestinal pathogenesis. It is protective as neutropenic mice develop significantly larger abscesses and lack the inflammatory ring of containment that is characteristic of these early lesions (Seydel et al., 1997b). It is also deleterious because neutrophil degranulation increases liver cell destruction (Salata and Ravdin, 1986). 3.2.3. Lesion expansion and granuloma formation As liver lesions progress immune cell infiltrates around the lesions alter. The second wave of cells is marked by an influx of macrophages that coincides with granuloma development. Granulomas are hallmarks of Th2 responses, as Th2 cytokines IL4 and IL-13 induce alternatively activated macrophages, which drive the formation of granulomas (Gordon, 2003; Wilson et al., 2007). IL-4 and IL-13 up-regulate macrophage Arginase-1 expression, which depletes intacellular L-arginine required for NO production and promotes arginase-dependent formation of Lornithine which ultimately leads to collagen production and fibroblast proliferation (Gordon, 2003). In the center of liver abscesses dense necrotic masses develop. Trophozoites aggregate at the inner side of granuloma walls and lyse fibrous material and any cells they encounter (Chadee and Meerovitch, 1985). In this manner trophozoites disseminate to adjacent locations and form multiple granulomas. Eventually these individual lesions coalesce into a single large mass enclosed by a thick fibrous wall, which is characteristic of the lesions found in human ALA. 3.2.3.1. Macrophages: Suppression. Although liver infections are dominated by a Th2 response, IFN-c production and iNOS expressing macrophages seem to play an important role in limiting abscess growth. Mice with a targeted iNOS deletion develop significantly larger abscesses than their wild-type counterparts (Seydel et al., 2000). Disruptions in the IFN-c receptor renders mice more susceptible to ALA and IFN-c neutralization in female mice, which are less susceptible than males to ALA, increases their susceptibility and severity of disease (Lotter et al., 2006). Large amounts of IFN-c are likely produced during the innate liver response by iNKT cells (as EhLPPG induces IFN-c production by iNKT cells) and other cellular sources, which activate macrophages to release NO into infected tissue. NO combined with toxic products from the oxidative burst then kills most, if not all liver trophozoites. However, the chronic stage of amebic liver disease is marked by defective cell-mediated immunity, particularly the inability of macrophages within abscesses to elicit a classical phenotype (high iNOS expression and NO production). In animal models of the disease, liver granuloma macrophages are refractive to IFN-c and LPS stimulation, do not produce inflammatory cytokines and are unable to kill trophozoites (Denis and Chadee, 1988; Wang et al., 1994). This suppression is local and is directly caused by the parasite, as macrophages at sites distant to liver lesions are unaffected

(Denis and Chadee, 1988; Wang et al., 1994). Moreover, trophozoite secretory components added to naive macrophages in vitro suppresses macrophage response towards LPS and/or IFN-c (Wang et al., 1994). Deactivated/suppressed macrophages are a distinct phenotype from alternative macrophages (Gordon, 2003). Alternative macrophages (i.e. activated by Th2 cytokines IL-4 and IL-13) not only promote wound repair/granulomas, but also express MHC II, are phagocytic and are highly capable of antigen presentation (Gordon, 2003). This is not the case with macrophages located within liver abscesses, where secreted ameba components decrease MHC II expression through a PGE2-dependent mechanism (Wang and Chadee, 1995). Inhibition of macrophage PGE2 synthesis can partially recover MHC II expression and TNF-a expression (Wang et al., 1994), which is also down-regulated. However, inhibition of PGE2 synthesis does not recover iNOS expression or amebicidal activity in trophozoite-deactivated macrophages. Other inhibitory pathways might therefore be exploited by the parasite, or given that E. histolytica synthesizes and secretes PGE2 (Dey et al., 2003), a continuous source of parasite-derived PGE2 may prevent iNOS expression and full recovery of MHC II and TNF-a, possibly through a concentration dependent-effect of PGE2. 3.2.3.2. T cell: Suppression. Beyond macrophage deactivation, suppression is also observed in T cells. An intriguing feature of human ALA is that recently recovered patients have skewed CD8+:CD4+ T cells ratios and abnormal proliferative responses to T cell mitogen concavalin A (ConA) (Salata et al., 1986). Their total T cell counts are normal, however, they have markedly increased and decreased numbers of CD8+ and CD4+ T cells, respectively. Most patients recover a normal CD8+:CD4+ ratio by one year post-infection, and the degree to which the ratio is restored correlates positively with a ConA proliferative response (Salata et al., 1986). T cell immunosuppression also occurs in animals with ALA. Insufficient levels of IL-2, the primary T cell growth factor, partially explain why serum from infected animals suppresses naive T cell proliferation to ConA (Campbell et al., 1999). E. histolytica-infected serum may contain factors which inhibit IL-2 production or soluble IL-2 receptors that consume released IL-2, as IL-2 supplementation partially recovers T cell proliferation in the presence of infected serum. The effect, however, is independent of PGE2, which mediates E. histolytica-induced macrophage suppression (Campbell et al., 1999). In humans, sera of recently cured ALA patients elicit this same suppression of T cell proliferation to E. histolytica antigens. In the presence of nonimmune sera lymphocytes from ALA patients proliferate robustly and produce IFN-c, whereas in the presence of immune sera proliferation is drastically impaired and little to no IFN-c is produced (Salata et al., 1990). Removing amebic antibodies from immune sera does not reverse the effect, which further supports the notion that immune sera contains factors that repress T cell responses. In addition to serum-mediated suppression, T cells from ALA animals also have defective proliferative responses in the presence of normal serum (Campbell et al., 1999). A mechanism for this has not been provided, and may be dependent on a number of molecules and processes. Together these data suggest that E. histolytica triggers development of Treg populations during chronic phases of disease that repress the development of responder T cells. Tregs are potent inhibitors of T cell proliferation and their mechanisms of suppression are many. Many studies have demonstrated Treg suppression occurs by repressing IL-2 gene expression and thereby IL-2 production by non-regulatory T cells (reviewed in Shevach, 2009). There are a number of cytokines with potent suppressive activity. IL-10 inhibits proliferative responses by directly inhibiting IL-2 production in CD4+ T cells and TGF-b can inhibit T cell proliferation and cytokine production (Roncarolo et al., 2006). Besides IL-10 and TGF-b, recently identified IL-35 is another suppressive cytokine

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candidate produced by Tregs. IL-35 belongs to the IL-12 cytokine family and is comprised of IL-12p35 (which pairs with IL-10p40 to form Th1 inducing IL-12) and Epstein–Barr virus-induced gene 3 (Ebi3, which pairs with IL-12p38 to form IL-27). Both these genes are up-regulated in actively suppressing Tregs and Tregs of mice deficient in either of these genes are unable to control proliferation and prevent inflammatory bowel disease in vivo (Shevach, 2009). Presently, there are no neutralizing antibodies to IL-35 nor is its receptor(s) known, so it is not currently possible to assess its functions directly. Several types of inducible Tregs are known, including Tr1 cells which produce large amounts of IL-10, TGF-bproducing Th3 cells and inducible Foxp3+ T cells (Faria and Weiner, 2005; Belkaid and Oldenhove, 2008). These cells and their mechanisms of suppression during chronic amebiasis remain to be identified. The consequences of Treg function can be broadly categorized into induction of cell cycle arrest, apoptosis and inhibition of pro-inflammatory cytokines (Shevach, 2009). How these cells might function in vivo during chronic stages of E. histolytica infections would be of immense interest. Human data and in vivo models indicate (though it has never been established) that the parasite skews the T cell response not only to Th2 cells, but also towards Tregs. Thus, the Th1/Th2 paradigm of amebiasis may need to be re-evaluated to include regulatory T cells. It would be interesting to investigate whether E. histolytica-induced Tregs would block Th1 differentiation that enables the host to clear/prevent chronic infections. If the parasite induces a regulatory T cell response, this would potentially have critical consequences for vaccination strategies; vaccine candidates would need to be carefully assessed for Treg induction that might be detrimental to the host during invasive E. histolytica infections.

4. Conclusions Both the innate and adaptive arms of the immune response participate in removing invasive E. histolytica. Invasion starts when amebae deplete the mucus layer and invade the colonic epithelium. In some cases, the parasite migrates into the lamina propria and submucosa and causes extensive ulceration. Once amebae subverts the mucosal barrier they may enter portal circulation and be carried to extraintestinal sites to establish extraintestinal lesions. Upon detecting intestinal invasion, the host mounts a rapid inflammatory response. Inflammatory signals are released that recruit an inflammatory cell infiltrate. Neutrophils are the first to arrive and are critical for containing the infection, but also inflict extensive tissue damage that exacerbates intestinal injury. It appears that in most situations an inflammatory response sufficiently controls the parasite, as amebiasis usually does not progress beyond dysentery and diarrhea. In patients with amebic colitis and ALA the parasite employs multiple strategies that allow it to successfully subvert the immune response and establish a chronic infection. In such cases the host develops a defective adaptive response incapable of clearing the parasite.

5. Future directions Over the past 30 years a picture of E. histolytica immunopathogenesis has emerged. However, there is still much to discover. E. histolytica is a facultative pathogen and its relationship with the host is complex. Efforts to date have not revealed why most individuals develop commensal infections, and why during colonic invasion most only succumb to an episode of diarrhea and dysentery. Fundamental questions about the host immune response and parasite virulence remain to be answered.

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Although 90% of E. histolytica infections result in harmless colonization of the colon, surprisingly we know little about how this interaction develops. A molecular understanding of the intestinal immune compartment’s normal reaction to E. histolytica will provide a framework to explain how a commensal interaction with the host is lost or not established. Host genetics seem to play a role, but there are a number of other non-mutually exclusive possibilities. For example nutritional status of the host and on-going immune responses to other pathogens are likely to influence the outcomes of infections. Evidence suggests that parasite genetics are also important. Though many candidate virulence factors have been identified, we still do not have a defined picture of which ones are required for intestinal and extraintestinal invasion, nor for many of these virulence factors, do we know the cellular and molecular mechanisms governing their effects (Table 1). The field of amebiasis has a wealth of knowledge to draw from. Our understanding is rapidly expanding of how the gastrointestinal tract achieves tolerance with the commensal flora, while still retaining the ability to detect and respond to pathogens. Many of these concepts will be applicable to E. histolytica and can shed light on the genesis of disease. Currently a major goal of E. histolytica research is to develop a vaccine for E. histolytica. An understanding of immune mechanisms not only during invasive infections, but also commensal infections will aid development of vaccination strategies. Acknowledgments Dr. Chadee is a Canada Research Chair (Tier 1) in Gastrointestinal Inflammation and his research is supported by grants from the Natural Sciences and Engineering Research Council of Canada, the Canadian Institute for Health Research, the Canadian Foundation for Innovation, the Crohn’s and Colitis Foundation of Canada, and the Canadian Association of Gastroenterology-Astra Zeneca-CIHR Research and Fellowship Awards. L. Mortimer is supported by Achievers in Medicine Award, Dean’s Entrance Scholarship (DES) and a FGS award. References Abd-Alla, M.D., Jackson, T.F., Rogers, T., Reddy, S., Ravdin, J.I., 2006. Mucosal immunity to asymptomatic Entamoeba histolytica and Entamoeba dispar infection is associated with a peak intestinal anti-lectin immunoglobulin A antibody response. Infection and Immunity 74, 3897–3903. Abreu, M.T., Vora, P., Faure, E., Thomas, L.S., Arnold, E.T., Arditi, M., 2001. Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. Journal of Immunology 167, 1609– 1616. Adams, E.B., MacLeod, I.N., 1977a. Invasive amebiasis I. Amebic dysentery and its complications. Medicine 56, 315–323. Adams, E.B., MacLeod, I.N., 1977b. Invasive amebiasis II. Amebic liver abscess and its complications. Medicine 56, 325–334. Ali, I.K., Utpal Mondal, U., Roy, S., Haque, R., Petri Jr., W.A., Clark, C.G., 2007. Evidence for a link between parasite genotype and outcome of infection with Entamoeba histolytica. Journal of Clinical Microbiology 45, 285–289. Ali, I.K., Solaymani-Mohammadi, S., Akhter, J., Roy, S., Gorrini, C., Calderaro, A., Parker, S.K., Haque, R., Petri Jr., W.A., Clark, C.G., 2008. Tissue invasion by Entamoeba histolytica: evidence of genetic selection and/or DNA reorganization events in organ tropism. PLoS Neglected Tropical Diseases 2, e219. Annacker, O., Coombes, J.L., Malmstrom, V., Uhlig, H.H., Bourne, T., JohanssonLindbom, B., Agace, W.W., Parker, C.M., Powrie, F., 2005. Essential role for CD103 in the T-cell-mediated regulation of experimental colitis. Journal of Experimental Medicine 202, 1051–1061. Artis, D., 2008. Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. Nature Reviews Immunology 8, 411–420. Asgharpour, A., Gilchrist, C., Baba, D., Hamano, S., Houpt, E., 2005. Resistance to intestinal Entamoeba histolytica infection is conferred by innate immunity and Gr-1+ Cells. Infection and Immunity 73, 4522–4529. Ayeh-Kumi, P.F., Ali, I.M., Lockhart, L.A., Gilchrist, C.A., Petri Jr., W.A., Haque, R., 2001. Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene. Experimental Parasitology 99, 80–88.

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